The binding of von Willebrand factor (vWF) to collagen type I and type III is the essential first step in normal hemostasis and in pathogenic thrombosis leading to stroke and myocardial infarction. So far the molecular interactions between the two molecules are poorly understood. While several collagen receptors are involved in the activation of platelets during thrombosis, the binding of vWF to collagen is required for anchoring platelets at the site of rupture of blood vessel wall under flow conditions. Our long-term goal is to understand how collagen interacts with the different collagen receptors on platelet and thereby modulates thrombosis. The objective of this work is to investigate molecular interactions of collagen with vWF. Both collagen and the mature vWF form multimeric molecular complexes. The A3 domain of vWF (vWF-A3) has been identified as the collagen binding domain, and we have successfully expressed the vWF-A3 in E. coli. Still, the large size and the unique rope-like structure of collagen triple helix make the investigation of specific molecular interactions between the two molecules challenging. We hypothesized that there are multiple vWF binding sites on collagen that are heterogeneous in binding capacity, and that the binding of vWF requires the joint action at the different sites. In order to test this hypothesis the proposed work will pursue the following specific aims: (1) to define the distribution of vWF-A3 binding sites in both type I and type III collagen using protein footprinting approach, and (2) to gain a quantitative understanding of the heterogeneous binding reactions between collagen and the vWF-A3 by quantitatively characterizing the thermodynamic and the kinetic properties of the binding interactions. The proposed work combines the strengths of the studies using the full-chain collagen and those using short synthetic peptides. The successful applications of the new approaches will lead to both detailed and general understanding of the binding reactions between collagen and vWF. The related knowledge will set up the stage for the further investigation of the molecular basis of the binding reaction and of the mode of action of the multiple binding sites during thrombosis. The collagen-vWF interaction represents a good target for the development of new and novel interventions for effective and safe control of thrombosis by inhibiting the process at its root.